TWEAK receptor

ABSTRACT

The present invention provides the TWEAK receptor and methods for identifying and using agonists and antagonists of the TWEAK receptor. In particular, the invention provides methods of screening for agonists and antagonists and for treating diseases or conditions mediated by angiogenesis, such as solid tumors and vascular deficiencies of cardiac or peripheral tissue.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/172,878, filed 20 Dec. 1999, and U.S. Provisional ApplicationSer. No. 60/203,347, filed 10 May 2000, both of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to the discovery of the functionalreceptor (TWEAKR) for the TWEAK protein. More particularly, theinvention relates to the use of TWEAKR agonists and antagonists inmethods of treatment, and to screening methods based on TWEAKR and theTWEAK-TWEAKR interaction.

BACKGROUND OF THE INVENTION

A. Angiogenesis

Angiogenesis is a multi-step developmental process that results in theformation of new blood vessels off of existing vessels. This spatiallyand temporally regulated process involves loosening of matrix contactsand support cell interactions in the existing vessels by proteases,followed by coordinated movement, morphological alteration, andproliferation of the smooth muscle and endothelial cells of the existingvessel. The nascent cells then extend into the target tissue followed bycell-cell interactions in which the endothelial cells form tubes whichthe smooth muscle cells surround. In a coordinated fashion,extracellular matrix proteins of the vessel are secreted andperi-endothelial support cells are recruited to support and maintainstructural integrity (see, e.g., Daniel et al., Ann. Rev. Physiol.2000(62):649, 2000). Angiogenesis plays important roles in both normaland pathological physiology.

Under normal physiological conditions, angiogenesis is involved in fetaland embryonic development, wound healing, organ regeneration, and femalereproductive remodeling processes including formation of theendometrium, corpus luteum, and placenta. Angiogenesis is stringentlyregulated under normal conditions, especially in adult animals, andperturbation of the regulatory controls can lead to pathologicalangiogenesis.

Pathological angiogenesis has been implicated in the manifestationand/or progression of inflammatory diseases, certain eye disorders, andcancer. In particular, several lines of evidence support the conceptthat angiogenesis is essential for the growth and persistence of solidtumors and their metastases (see, e.g., Folkman, N. Engl. J. Med.285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991). Angiogenesisinhibitors are therefore useful for the prevention (e.g., treatment ofpremalignant conditions), intervention (e.g., treatment of smalltumors), and regression (e.g., treatment of large tumors) of cancers(see, e.g., Bergers et al., Science 284:808, 1999).

There is a need for additional compositions and methods of modulatingangiogenesis for the prevention, abrogation, and mitigation of disease.

B. TWEAK

The TWEAK protein, which has also been called TREPA and Apo3L, is amember of the tumor necrosis factor (TNF) family and is expressed in awide variety of human tissues (Chicheportiche et al., J. Biol. Chem.,272(51):32401, 1997; see also Wiley, PCT Publication No. WO 98/35061, 13Aug. 1998). Like most TNF family members, TWEAK is a Type II membraneprotein with an extracellular C-terminal domain. Although TWEAK wasoriginally described as a weak inducer of apoptosis, this induction ofcell death was later shown to be indirect (Schneider et al., Eur. J.Immunol. 29:1785, 1999).

Lynch et al. demonstrated that TWEAK directly induces endothelial cellproliferation and angiogenesis (J. Biol. Chem., 274(13):8455, 1999).Picomolar concentrations of recombinant soluble TWEAK induceproliferation in multiple endothelial cell lines and in aortic smoothmuscle cells, and reduce the requirement for serum and growth factors inculture. Moreover, TWEAK induces a strong angiogenic response in a ratcorneal pocket assay. Since TNF family members initiate biologicalresponses by signaling through members of the TNF receptor family, therehas been great interest in identifying and characterizing the TWEAKreceptor.

Marsters et al. reported that TWEAK binds to and signals through adeath-domain containing receptor known variously as DR3, Apo3, WSL-1,TRAMP, or LARD (Marsters et al., Current Biology 8(9):525, 1998).Schneider et al., however, showed that TWEAK binds to and signals inKym-1 cells but that Kym-1 cells do not express the receptor DR3(Schneider et al., Eur. J. Immunol. 29:1785, 1999). These resultssuggest the existence of a yet to be identified TWEAK receptor.

Because TWEAK induces angiogenesis in vivo, there is a particular needto identify the major functional TWEAK receptor. Once identified, theTWEAK receptor may be used to screen for and develop TWEAK receptoragonists and antagonists for the modulation of angiogenesis and thetreatment of human disease.

SUMMARY OF THE INVENTION

The present invention is based upon the identification and biologicalcharacterization of the major functional TWEAK receptor. As describedbelow, cDNA encoding the TWEAK receptor was molecularly cloned from ahuman endothelial cell expression library.

Although DNA and deduced amino acid sequences corresponding to the TWEAKreceptor identified herein have been reported (see, e.g., Kato et al.,PCT Publication No. WO 98/55508, 10 Dec. 1998 and Incyte, PCTPublication No. WO 99/61471, 2 Dec. 1999), it was not heretoforeappreciated that these sequences encode a receptor for TWEAK or that theencoded polypeptide is involved in modulating angiogenesis. Similarly,investigators have recently claimed methods of making and using TWEAKreceptor antagonists to treat immunological disorders, but withoutidentifying the major TWEAK receptor or its role in angiogenesis(Rennert, PCT Publication No. WO 00/42073, 20 Jul. 2000). Thesedeficiencies have been addressed, as described herein, by identificationof the major TWEAK receptor (TWEAKR) and characterization of itsbiological activities. The identification of TWEAKR has led to thedevelopment of compositions for the modulation of angiogenesis, and alsoprovides screening tools for the identification of diagnostics andtherapeutics.

The invention provides methods of modulating angiogenesis in a mammal inneed of such treatment comprising administering atherapeutically-effective amount of a composition comprising a TWEAKreceptor antagonist or TWEAK receptor agonist. The compositionpreferably comprises a pharmaceutically acceptable carrier and themammal is preferably a human.

In some more preferred embodiments the composition inhibits angiogenesisand comprises a TWEAK receptor antagonist, such as a soluble TWEAKreceptor fragment, an antagonistic antibody, or an antagonist thatdisrupts the interaction between the TWEAK receptor and a TRAF molecule.In some most preferred embodiments the antagonist comprises amino acids28-79 of SEQ ID NO:7 or amino acids 28-309 of SEQ ID NO:7. The TWEAKreceptor antagonists are preferably used to treat a mammal that has adisease or condition mediated by angiogenesis, more preferably a diseaseor condition characterized by ocular neovascularization or a solidtumor. In some embodiments, the mammal is further treated with radiationor with a second chemotherapeutic agent.

In some more preferred embodiments the composition promotes angiogenesisand comprises a TWEAK receptor agonist, such as an agonistic antibody.The TWEAK receptor agonists are preferably used to treat avascularization deficiency in cardiac or peripheral tissue, to enhancewound healing or organ transplantation, or in conjunction with bypasssurgery or angioplasty.

The invention also provides antagonists comprising a soluble TWEAKreceptor fragment for use in medicine, preferably comprising amino acids28-79 of SEQ ID NO:7 or amino acids 28-309 of SEQ ID NO:7, as well asnucleic acids encoding soluble TWEAK receptor fragments. And theinvention provides for the use of a composition comprising a TWEAKreceptor antagonist or TWEAK receptor agonist for the preparation of amedicament for modulating angiogenesis in a mammal in need of suchtreatment.

The invention further provides methods of identifying a compound that iscapable of modulating angiogenesis, including: (a) identifying a testcompound that binds to a TWEAK receptor extracellular domain, whereinthe test compound is not TWEAK; (b) identifying a test compound thataffects the interaction between a TWEAK and a TWEAK receptor; and (c)identifying a test compound that modulates the interaction between aTWEAK receptor and a TRAF. The invention encompasses compoundsidentified according to these methods.

The invention also provides a method for targeting a detectable label orchemotherapeutic to vascular tissue comprising contacting the vasculartissue with an antibody that binds TWEAK receptor. In some preferredembodiments the antibody is conjugated to a radioisotope,chemiluminescent or fluorescent compound, or enzyme. In some preferredembodiments the antibody is conjugated to a cytotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of the human and murine TWEAK receptorpolypeptide sequences. The top sequence is the murine TWEAK receptorpolypeptide (SEQ ID NO:5), and the bottom sequence is the human TWEAKreceptor polypeptide (SEQ ID NO:4).

FIG. 2 shows the effect of TWEAKR-Fc on PMA-induced HRMEC wound closure.

FIG. 3 shows the effect of TWEAKR-Fc on EGF-induced HRMEC wound closure.

FIG. 4 shows the effect of human TWEAKR-Fc on TWEAK-induced (100 ng/ml)HUVEC proliferation.

FIG. 5 shows the effect of human TWEAKR-Fc on FGF-2-induced (10 ng/mi)HUVEC proliferation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the TWEAK receptor and methods foridentifying and using agonists and antagonists of the TWEAK receptor.The invention provides methods of screening for agonists and antagonistsand for treating diseases or conditions mediated by angiogenesis.

A. Abbreviations and Terminology Used in the Specification

“4-1BB” and “4-1BB ligand” (4-1BB-L) are polypeptides described, interalia, in U.S. Pat. No. 5,674,704, including soluble forms thereof.

“bFGF” is basic fibroblast growth factor.

“BSA” is bovine serum albumin.

“CD40 ligand” (CD40L) is a polypeptide described, inter alia, in U.S.Pat. No. 5,716,805, including soluble forms thereof.

“CHO” is a Chinese hamster ovary cell line.

“DMEM” is Dulbecco's Modified eagle Medium, a commercially availablecell culture medium.

“ELISA” is Enzyme-Linked Immunosorbent Assay.

“Flt3L” is Flt3 ligand, a polypeptide described, inter alia, in U.S.Pat. No. 5,554,512, including soluble forms thereof.

“HRMEC” are primary human renal microvascular endothelial cells.

“HUVEC” is a line of human umbilical vein endothelial cells.

“PBS” is phosphate buffered saline.

“PMA” is phorbol 12-myristate-13-acetate.

“RTKs” are receptor tyrosine kinases.

“Tek,” which has also been called Tie2 and ork, is an RTK that ispredominantly expressed in vascular endothelium. The molecular cloningof human Tek (ork) has been described by Ziegler, U.S. Pat. No.5,447,860. “Tek antagonists” are described, inter alia, in Cerretti etal., PCT Publication No.

WO 00/75323, 14 Dec. 2000.

“TNFR” is a tumor necrosis factor receptor, including soluble formsthereof. “TNFR/Fc” is a tumor necrosis factor receptor-Fc fusionpolypeptide.

“TRAIL” is TNF-related apoptosis-inducing ligand, a type IItransmembrane polypeptide in the TNF family described, inter alia, inU.S. Pat. No. 5,763,223, including soluble forms thereof.

“VEGF” is vascular endothelial growth factor, also known as VPF orvascular permeability factor.

B. Soluble TWEAK Receptor Polypeptides

As described in the examples below, the native human TWEAK receptor cDNAhas the sequence SEQ ID NO:3, which encodes a 129 residue polypeptide(SEQ ID NO:4). Examination of the DNA sequence predicts a polypeptidehaving an approximately 78 amino acid extracellular domain (residues1-78 of SEQ ID NO:4, including the signal peptide), an approximately 23amino acid transmembrane domain (residues 79-101 of SEQ ID NO:4), and anapproximately 28 amino acid intracellular domain (residues 102-129 ofSEQ ID NO:4). The TWEAK receptor sequence has also been reported by Katoet al., PCT Publication No. WO 98/55508, 10 Dec. 1998 and by Incyte, PCTPublication No. WO 99/61471, 2 Dec. 1999. As used herein, “TWEAKR”includes polypeptides having these sequences, and in particularcomprising amino acids 28-79 of SEQ ID NO:7, as well as naturallyoccurring variants thereof.

In one aspect of the invention, a soluble TWEAK receptor fragment isused as a TWEAKR antagonist to inhibit angiogenesis and/or to inhibitthe binding of TWEAK ligand to TWEAKR.

Soluble polypeptides are capable of being secreted from the cells inwhich they are expressed. The use of soluble forms of polypeptides isadvantageous for certain applications. Purification of the polypeptidesfrom recombinant host cells is facilitated since the polypeptides aresecreted, and soluble proteins are generally suited for parenteraladministration. A secreted soluble polypeptide may be identified (anddistinguished from its non-soluble membrane-bound counterparts) byseparating intact cells which express the desired polypeptide from theculture medium, e.g., by centrifugation, and assaying the medium(supernatant) for the presence of the desired polypeptide. The presenceof the desired polypeptide in the medium indicates that the polypeptidewas secreted from the cells and thus is a soluble form of thepolypeptide. Soluble polypeptides may be prepared by any of a number ofconventional techniques. A DNA sequence encoding a desired solublepolypeptide may be subcloned into an expression vector for production ofthe polypeptide, or the desired encoding DNA fragment may be chemicallysynthesized.

Soluble TWEAKR polypeptides comprise all or part of the TWEAKRextracellular domain, but generally lack the transmembrane domain thatwould cause retention of the polypeptide at the cell surface. Solublepolypeptides may include part of the transmembrane domain or all or partof the cytoplasmic domain as long as the polypeptide is secreted fromthe cell in which it is produced. Soluble TWEAKR polypeptidesadvantageously comprise a native or heterologous signal peptide wheninitially synthesized, to promote secretion from the cell, but thesignal sequence is cleaved upon secretion. The term “TWEAKRextracellular domain” is intended to encompass all or part of the nativeTWEAKR extracellular domain, as well as related forms including but notlimited to: (a) fragments, (b) variants, (c) derivatives, and (d) fusionpolypeptides. The ability of these related forms to inhibit angiogenesisor other TWEAKR-mediated responses may be determined in vitro or invivo, using methods such as those exemplified below or using otherassays known in the art. Examples of soluble TWEAKR polypeptides areprovided below. In some embodiments of the present invention amultimeric form of a soluble TWEAKR polypeptide (“soluble TWEAKRmultimer”) is used as an antagonist to block the binding of TWEAK toTWEAKR, to inhibit angiogenesis or other TWEAKR-mediated responses.

Soluble TWEAKR multimers are covalently-linked or non-covalently-linkedmultimers, including dimers, trimers, or higher multimers. Multimers maybe linked by disulfide bonds formed between cysteine residues ondifferent soluble TWEAKR polypeptides. One embodiment of the inventionis directed to multimers comprising multiple soluble TWEAKR polypeptidesjoined via covalent or non-covalent interactions between peptidemoieties fused to the soluble TWEAKR polypeptides. Such peptides may bepeptide linkers (spacers), or peptides that have the property ofpromoting multimerization. Leucine zippers and certain polypeptidesderived from antibodies are among the peptides that can promotemultimerization of soluble TWEAKR polypeptides attached thereto, asdescribed in more detail below. In particular embodiments, the multimerscomprise from two to four soluble TWEAKR polypeptides.

In some embodiments, a soluble TWEAKR multimer is prepared usingpolypeptides derived from immunoglobulins. Preparation of fusionproteins comprising certain heterologous polypeptides fused to variousportions of antibody-derived polypeptides (including the Fc domain) hasbeen described, e.g., by Ashkenazi et al. (Proc. Natl. Acad. Sci. USA88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh andAruffo (“Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992).

One preferred embodiment of the present invention is directed to aTWEAKR-Fc dimer comprising two fusion proteins created by fusing solubleTWEAKR to an Fc polypeptide. A gene fusion encoding the TWEAKR-Fc fusionprotein is inserted into an appropriate expression vector. TWEAKR-Fcfusion proteins are expressed in host cells transformed with therecombinant expression vector, and allowed to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fc moieties to yield divalent soluble TWEAKR. The term “Fcpolypeptide” as used herein includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization are also included.

One suitable Fc polypeptide, described in PCT application WO 93/10151,is a single chain polypeptide extending from the N-terminal hinge regionto the native C-terminus of the Fc region of a human IgGI antibody.Another useful Fc polypeptide is the Fc mutein described in U.S. Pat.No. 5,457,035 and by Baum et al., EMBO J. 13:3992, 1994. The amino acidsequence of this mutein is identical to that of the native Fc sequencepresented in WO 93/10151, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors. Fusion polypeptides comprising Fcmoieties, and multimers formed therefrom, offer an advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns, and Fc fusion polypeptides may provide a longer in vivo halflife, which is useful in therapeutic applications, than unmodifiedpolypeptides.

In other embodiments, a soluble TWEAKR polypeptide may be substitutedfor the variable portion of an antibody heavy or light chain. If fusionproteins are made with both heavy and light chains of an antibody, it ispossible to form a soluble TWEAKR multimer with as many as four solubleTWEAKR polypeptides.

Alternatively, the soluble TWEAKR multimer is a fusion proteincomprising multiple soluble TWEAKR polypeptides, with or without peptidelinkers (spacers), or peptides that have the property of promotingmultimerization. Among the suitable peptide linkers are those describedin U.S. Pat. Nos. 4,751,180, 4,935,233, and 5,073,627. A DNA sequenceencoding a desired peptide linker may be inserted between, and in thesame reading frame as, the DNA sequences encoding TWEAKR, usingconventional techniques known in the art. For example, a chemicallysynthesized oligonucleotide encoding the linker may be ligated betweensequences encoding soluble TWEAKR. In particular embodiments, a fusionprotein comprises from two to four soluble TWEAKR polypeptides,separated by peptide linkers.

Another method for preparing soluble TWEAKR multimers involves use of aleucine zipper domain. Leucine zipper domains are peptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, 1988), and have since been found in a varietyof different proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.Examples of leucine zipper domains suitable for producing solublemultimeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. FEBS Lett. 344:191, 1994. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al.,Semin. Immunol. 6:267, 1994. Recombinant fusion proteins comprising asoluble TWEAKR polypeptide fused to a leucine zipper peptide areexpressed in suitable host cells, and the soluble TWEAKR multimer thatforms is recovered from the culture supernatant.

For some applications, the soluble TWEAKR multimers of the presentinvention are believed to provide certain advantages over the use ofmonomeric forms. Fc fusion polypeptides, for example, typically exhibitan increased in vivo half life as compared to an unmodified polypeptide.

The present invention encompasses the use of various forms of solubleTWEAKR multimers that retain the ability to inhibit angiogenesis orother TWEAKR-mediated responses. The term “soluble TWEAKR multimer” isintended to encompass multimers containing all or part of the nativeTWEAKR extracellular domain, as well as related forms including, but notlimited to, multimers of: (a) fragments, (b) variants, (c) derivatives,and (d) fusion polypeptides of soluble TWEAKR. The ability of theserelated forms to inhibit angiogenesis or other TWEAKR-mediated responsesmay be determined in vitro or in vivo, using methods such as thoseexemplified in the examples or using other assays known in the art.

Among the soluble TWEAKR polypeptides and soluble TWEAKR multimersuseful in practicing the present invention are TWEAKR variants thatretain the ability to bind ligand and/or inhibit angiogenesis or otherTWEAKR-mediated responses. Such TWEAKR variants include polypeptidesthat are substantially homologous to native TWEAKR, but which have anamino acid sequence different from that of a native TWEAKR because ofone or more deletions, insertions or substitutions. Particularembodiments include, but are not limited to, TWEAKR polypeptides thatcomprise from one to ten deletions, insertions or substitutions of aminoacid residues, when compared to a native TWEAKR sequence. Included asvariants of TWEAKR polypeptides are those variants that are naturallyoccurring, such as allelic forms and alternatively spliced forms, aswell as variants that have been constructed by modifying the amino acidsequence of a TWEAKR polypeptide or the nucleotide sequence of a nucleicacid encoding a TWEAKR polypeptide.

Generally, substitutions for one or more amino acids present in thenative polypeptide should be made conservatively. Examples ofconservative substitutions include substitution of amino acids outsideof the active domain(s), and substitution of amino acids that do notalter the secondary and/or tertiary structure of TWEAKR. Additionalexamples include substituting one aliphatic residue for another, such asIle, Val, Leu, or Ala for one another, or substitutions of one polarresidue for another, such as between Lys and Arg; Glu and Asp; or Glnand Asn, or substitutions of one aromatic residue for another, such asPhe, Trp, or Tyr for one another. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity characteristics, are known in the art.

In some preferred embodiments the TWEAKR variant is at least about 70%identical in amino acid sequence to the amino acid sequence of nativeTWEAKR; in some preferred embodiments the TWEAKR variant is at leastabout 80% identical in amino acid sequence to the amino acid sequence ofnative TWEAKR. In some more preferred embodiments the TWEAKR variant isat least about 90% identical in amino acid sequence to the amino acidsequence of native TWEAKR; in some more preferred embodiments the TWEAKRvariant is at least about 95% identical in amino acid sequence to theamino acid sequence of native TWEAKR. In some most preferred embodimentsthe TWEAKR variant is at least about 98% identical in amino acidsequence to the amino acid sequence of native TWEAKR; in some mostpreferred embodiments the TWEAKR variant is at least about 99% identicalin amino acid sequence to the amino acid sequence of native TWEAKR.Percent identity, in the case of both polypeptides and nucleic acids,may be determined by visual inspection. Percent identity may also bedetermined using the alignment method of Needleman and Wunsch (J. Mol.Biol. 48:443, 1970) as revised by Smith and Waterman (Adv. Appl. Math2:482, 1981. Preferably, percent identity is determined by using acomputer program, for example, the GAP computer program version 10.xavailable from the Genetics Computer Group (GCG; Madison, Wis., see alsoDevereux et al., Nucl. Acids Res. 12:387, 1984). The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp.353-358, 1979 for amino acids; (2) a penalty of30 (amino acids) or 50 (nucleotides) for each gap and an additional 1(amino acids) or 3 (nucleotides) penalty for each symbol in each gap;(3) no penalty for end gaps; and (4) no maximum penalty for long gaps.Other programs used by one skilled in the art of sequence comparison mayalso be used. For fragments of TWEAKR, the percent identity iscalculated based on that portion of TWEAKR that is present in thefragment.

The present invention further encompasses the use of soluble TWEAKRpolypeptides with or without associated native-pattern glycosylation.TWEAKR expressed in yeast or mammalian expression systems (e.g., COS-1or COS-7 cells) may be similar to or significantly different from anative TWEAKR polypeptide in molecular weight and glycosylation pattern,depending upon the choice of expression system. Expression of TWEAKRpolypeptides in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Different host cells may also processpolypeptides differentially, resulting in heterogeneous mixtures ofpolypeptides with variable N- or C-termini.

The primary amino acid structure of soluble TWEAKR polypeptides may bemodified to create derivatives by forming covalent or aggregativeconjugates with other chemical moieties, such as glycosyl groups,lipids, phosphate, acetyl groups and the like. Covalent derivatives ofTWEAKR may be prepared by linking particular functional groups to TWEAKRamino acid side chains or at the N-terminus or C-terminus of a TWEAKRpolypeptide.

Fusion polypeptides of soluble TWEAKR that are useful in practicing theinvention also include covalent or aggregative conjugates of a TWEAKRpolypeptide with other polypeptides added to provide novelpolyfunctional entities.

C. TWEAK Receptor Antibodies

One aspect of the present invention relates to the antigenic epitopes ofthe TWEAKR extracellular domain. Such epitopes are useful for raisingantibodies, and in particular the blocking monoclonal antibodiesdescribed in more detail below. Such epitopes or variants thereof can beproduced using techniques well known in the art such as solid-phasesynthesis, chemical or enzymatic cleavage of a polypeptide, or usingrecombinant DNA technology.

The claimed invention encompasses compositions and uses of antibodiesthat are immunoreactive with TWEAKR polypeptides. Such antibodies “bindspecifically” to TWEAKR polypeptides, meaning that they bind viaantigen-binding sites of the antibody as compared to non-specificbinding interactions. The terms “antibody” and “antibodies” are usedherein in their broadest sense, and include, without limitation, intactmonoclonal and polyclonal antibodies as well as fragments such as Fv,Fab, and F(ab′)2 fragments, single-chain antibodies such as scFv, andvarious chain combinations. The antibodies of the present invention arepreferably humanized, and more preferably human. The antibodies may beprepared using a variety of well-known methods including, withoutlimitation, immunization of animals having native or transgenic immunerepertoires, phage display, hybridoma and recombinant cell culture, andtransgenic plant and animal bioreactors.

Both polyclonal and monoclonal antibodies may be prepared byconventional techniques. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide, harvesting spleen cells from the immunizedanimal, fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells, and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies produced by hybridomas may be recovered byconventional techniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., “humanized” versions of antibodies originally producedin mice or other non-human species. A humanized antibody is anengineered antibody that typically comprises the variable region of anon-human (e.g., murine) antibody, or at least complementaritydetermining regions (CDRs) thereof, and the remaining immunoglobulinportions derived from a human antibody. Procedures for the production ofchimeric and further engineered monoclonal antibodies include thosedescribed in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winterand Harris (TIPS 14:139, May, 1993). Such humanized antibodies may beprepared by known techniques and offer the advantage of reducedimmunogenicity when the antibodies are administered to humans.

Procedures that have been developed for generating human antibodies innon-human animals may be employed in producing antibodies of the presentinvention. The antibodies may be partially human or preferablycompletely human. For example, transgenic mice into which geneticmaterial encoding one or more human immunoglobulin chains has beenintroduced may be employed. Such mice may be genetically altered in avariety of ways. The genetic manipulation may result in humanimmunoglobulin polypeptide chains replacing endogenous immunoglobulinchains in at least some, and preferably virtually all, antibodiesproduced by the animal upon immunization.

Mice in which one or more endogenous immunoglobulin genes have beeninactivated by various means have been prepared. Human immunoglobulingenes have been introduced into the mice to replace the inactivatedmouse genes. Antibodies produced in the animals incorporate humanimmunoglobulin polypeptide chains encoded by the human genetic materialintroduced into the animal. Examples of techniques for the productionand use of such transgenic animals to make antibodies (which aresometimes called “transgenic antibodies”) are described in U.S. Pat.Nos. 5,814,318, 5,569,825, and 5,545,806, which are incorporated byreference herein.

D. Inhibitory Antisense, Ribozyme, and Triple Helix Approaches

Modulation of angiogenesis in a tissue or group of cells may also beameliorated by decreasing the level of TWEAKR gene expression and/orTWEAK receptor-ligand interaction by using TWEAK receptor or ligand genesequences in conjunction with well-known antisense, gene “knock-out,”ribozyme and/or triple helix methods to decrease the level of TWEAKreceptor or ligand gene expression. Among the compounds that may exhibitthe ability to modulate the activity, expression or synthesis of theTWEAK receptor or ligand gene, including the ability to modulateangiogenesis, are antisense, ribozyme, and triple helix molecules. Suchmolecules may be designed to reduce or inhibit either unimpaired, or ifappropriate, mutant target gene activity. Techniques for the productionand use of such molecules are well known to those of skill in the art.

E. Recombinant Production of TWEAK Receptor Polypeptides

TWEAKR polypeptides, including soluble TWEAKR polypeptides, fragments,and fusion polypeptides, used in the present invention may be preparedusing a recombinant expression system. Host cells transformed with arecombinant expression vector (“recombinant host cells”) encoding theTWEAKR polypeptide are cultured under conditions that promote expressionof TWEAKR and the TWEAKR is recovered. TWEAKR polypeptides can also beproduced in transgenic plants or animals, or by chemical synthesis.

The invention encompasses nucleic acid molecules encoding the TWEAKRpolypeptides used in the invention, including: (a) nucleic acids thatencode residues 28-79 of SEQ ID NO:7 and fragments thereof that bindTWEAK; (b) nucleic acids that are at least 70%, 80%, 90%, 95%, 98%, or99% identical to a nucleic acid of (a), and which encode a polypeptidecapable of binding TWEAK; and (c) nucleic acids that hybridize atmoderate stringency to a nucleic acid of (a), and which encode apolypeptide capable of binding TWEAK.

Due to degeneracy of the genetic code, there can be considerablevariation in nucleotide sequences encoding the same amino acid sequence.Included as embodiments of the invention are nucleic acid sequencescapable of hybridizing under moderately stringent conditions (e.g.,prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization conditions of 50° C., 5×SSC, overnight) to the DNAsequences encoding TWEAKR. The skilled artisan can determine additionalcombinations of salt and temperature that constitute moderatehybridization stringency (see also, Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1982; and Ausubel, Current Protocols in Molecular Biology, Wileyand Sons, 1989 and later versions, which are incorporated herein byreference). Conditions of higher stringency include higher temperaturesfor hybridization and post-hybridization washes, and/or lower saltconcentration. Percent identity of nucleic acids may be determined usingthe methods described above for polypeptides, i.e., by methods includingvisual inspection and the use of computer programs such as GAP.

Any suitable expression system may be employed for the production ofrecombinant TWEAKR. Recombinant expression vectors include DNA encodinga TWEAKR polypeptide operably linked to suitable transcriptional andtranslational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Nucleotide sequencesare operably linked when the regulatory sequence functionally relates tothe TWEAKR DNA sequence. Thus, a promoter nucleotide sequence isoperably linked to a TWEAKR DNA sequence if the promoter nucleotidesequence controls the transcription of the TWEAKR DNA sequence. Examplesof regulatory sequences include transcriptional promoters, operators, orenhancers, an mRNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.A sequence encoding an appropriate signal peptide (native orheterologous) can be incorporated into expression vectors. A DNAsequence for a signal peptide (referred to by a variety of namesincluding secretory leader, leader peptide, or leader) may be fused inframe to the TWEAKR sequence so that the TWEAKR polypeptide is initiallytranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cells promotesextracellular secretion of the TWEAKR polypeptide. The signal peptide iscleaved from the TWEAKR polypeptide upon secretion of TWEAKR from thecell.

Suitable host cells for expression of TWEAKR polypeptides includeprokaryotes, yeast and higher eukaryotic cells, including insect andmammalian cells. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, insect, and mammalian cellular hosts aredescribed, for example, in Pouwels et al. Cloning Vectors: A LaboratoryManual, Elsevier, N.Y., 1985.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, TWEAKR polypeptides may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker gene(s). A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a TWEAKRDNA sequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

TWEAKR polypeptides may also be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phospho-glucose isomerase, and glucokinase. Other suitablevectors and promoters for use in yeast expression are further describedin Hitzeman, EPA-73,657. Another alternative is the glucose-repressibleADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674,1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectorsreplicable in both yeast and E. coli may be constructed by inserting DNAsequences from pBR322 for selection and replication in E. coli (Amp^(r)gene and origin of replication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof recombinant polypeptides. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl.Acad. Sci. USA 81 :5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Insect host cell culture systems also may be employed to expressrecombinant TWEAKR polypeptides, including soluble TWEAKR polypeptides.Bacculovirus systems for production of heterologous polypeptides ininsect cells are reviewed by Luckow and Summers, Bio/Technology 6:47,1988.

Mammalian cells are particularly preferred for use as host cells.Examples of suitable mammalian host cell lines include the COS-7 line ofmonkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981),L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary(CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and theCV1/EBNA cell line derived from the African green monkey kidney cellline CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821,1991). For the production of therapeutic polypeptides it is particularlyadvantageous to use a mammalian host cell line which has been adapted togrow in media that does not contain animal proteins.

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used totransfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413,1987). In addition, electroporation can be used to transfect mammaliancells using conventional procedures, such as those in Sambrook et al.Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold SpringHarbor Laboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B 11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216, 1980). A plasmidexpressing the DHFR cDNA can be introduced into strain DX-B 11, and onlycells that contain the plasmid can grow in the appropriate selectivemedia. Other examples of selectable markers that can be incorporatedinto an expression vector include cDNAs conferring resistance toantibiotics, such as G418 and hygromycin B. Cells harboring the vectorcan be selected on the basis of resistance to these compounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529-534) and thetripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras etal., J. Biol. Chem. 257:13475, 1982). The internal ribosome entry site(IRES) sequences of viral origin allows dicistronic mRNAs to betranslated efficiently (Oh and Sarnow, Current Opinion in Genetics andDevelopment 3:295, 1993; Ramesh et al., Nucleic Acids Research 24:2697,1996). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by Mosser et al., Biotechniques 22:150, 1997, and p2A5Idescribed by Morris et al., Animal Cell Technology, 1997, pp.529-534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335, 1989. Other expression vectors for use in mammalianhost cells can be constructed as disclosed by Okayama and Berg (Mol.Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are known inthe art.

Regarding signal peptides that may be employed in producing TWEAKRpolypeptides, the native TWEAKR signal peptide may used or it may bereplaced by a heterologous signal peptide or leader sequence, ifdesired. The choice of signal peptide or leader may depend on factorssuch as the type of host cells in which the recombinant TWEAKR is to beproduced. Examples of heterologous signal peptides that are functionalin mammalian host cells include the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846.

Using the techniques of recombinant DNA including mutagenesis and thepolymerase chain reaction (PCR), the skilled artisan can produce DNAsequences that encode TWEAKR polypeptides comprising various additionsor substitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences, including TWEAKR fragments,variants, derivatives, and fusion polypeptides.

Transgenic animals, including mice, goats, sheep, and pigs, andtransgenic plants, including tobacco, tomato, legumes, grasses, andgrains, may also be used as bioreactors for the production of TWEAKRpolypeptides, including soluble TWEAKR polypeptides. In the case oftransgenic animals, it is particularly advantageous to construct achimeric DNA including a TWEAKR coding sequence operably linked tocis-acting regulatory sequences that promote expression of the solubleTWEAKR in milk and/or other body fluids (see, e.g., U.S. Pat. No.5,843,705; U.S. Pat. No. 5,880,327). In the case of transgenic plants itis particularly advantageous to produce TWEAKR in a particular celltype, tissue, or organ (see, e.g., U.S. Pat. No. 5,639,947; U.S. Pat.No. 5,889,189).

The skilled artisan will recognize that the procedure for purifyingexpressed soluble TWEAKR polypeptides will vary according to the hostsystem employed, and whether or not the recombinant polypeptide issecreted. Soluble TWEAKR polypeptides may be purified using methodsknown in the art, including one or more concentration, salting-out, ionexchange, hydrophobic interaction, affinity purification, HPLC, or sizeexclusion chromatography steps. Fusion polypeptides comprising Fcmoieties (and multimers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

F. Methods of Treatment

Described below are methods and compositions employing the TWEAKreceptor or ligand, or the genes encoding the TWEAK receptor or ligand,to promote or suppress angiogenesis in a target tissue or group ofcells. The terms “treat,” “treating,” “treatment,” “therapy,”“therapeutic,” and the like are intended to include preventativetherapy, prophylactic therapy, ameliorative therapy, and curativetherapy.

The disclosed polypeptides, compositions, and methods are used toinhibit angiogenesis or other TWEAKR-mediated responses in a mammal inneed of such treatment. The term “TWEAKR-mediated response” includes anycellular, physiological, or other biological response that is caused atleast in part by the binding of TWEAK ligand to TWEAKR, or which may beinhibited or suppressed, in whole or in part, by blocking TWEAK frombinding to TWEAKR. The treatment is advantageously administered in orderto prevent the onset or the recurrence of a disease or conditionmediated by angiogenesis, or to treat a mammal that has a disease orcondition mediated by angiogenesis. Diseases and conditions mediated byangiogenesis include but are not limited to ocular disorders, malignantand metastatic conditions, and inflammatory diseases.

Among the ocular disorders that can be treated according to the presentinvention are eye diseases characterized by ocular neovascularizationincluding, but not limited to, diabetic retinopathy (a majorcomplication of diabetes), retinopathy of prematurity (this devastatingeye condition, that frequently leads to chronic vision problems andcarries a high risk of blindness, is a severe complication during thecare of premature infants), neovascular glaucoma, retinoblastoma,retrolental fibroplasia, rubeosis, uveitis, macular degeneration, andcorneal graft neovascularization. Other eye inflammatory diseases,ocular tumors, and diseases associated with choroidal or irisneovascularization can also be treated according to the presentinvention.

The present invention can also be used to treat malignant and metastaticconditions such as solid tumors. Solid tumors include both primary andmetastatic sarcomas and carcinomas.

The present invention can also be used to treat inflammatory diseasesincluding, but not limited to, arthritis, rheumatism, and psoriasis.

Other diseases and conditions that can be treated according to thepresent invention include benign tumors and preneoplastic conditions,myocardial angiogenesis, hemophilic joints, scleroderma, vascularadhesions, atherosclerotic plaque neovascularization, telangiectasia,and wound granulation.

Disease states that are angiogenic-dependent include coronary orperipheral atherosclerosis and ischemia of any tissue or organ,including the heart, liver, brain, and the like. These types of diseasescan be treated by compositions that promote angiogenesis.

In addition to polypeptides comprising a fragment of TWEAKRextracellular domain, soluble TWEAKR multimers, and antibodies that bindto the TWEAKR extracellular domain, other forms of TWEAKR antagonistscan also be administered to achieve a therapeutic effect. Examples ofother forms of TWEAKR antagonists include other antibodies such asantibodies against TWEAK, antisense nucleic acids, ribozymes, muteins,aptamers, and small molecules directed against TWEAKR or against TWEAK.

The methods according to the present invention can be tested in in vivoanimal models to confirm the desired prophylactic or therapeuticactivity, as well as to determine the optimal therapeutic dosage, priorto administration to humans.

The amount of a particular TWEAKR antagonist that will be effective in aparticular method of treatment depends upon age, type and severity ofthe condition to be treated, body weight, desired duration of treatment,method of administration, and other parameters. Effective dosages aredetermined by a physician or other qualified medical professional.Typical effective dosages are about 0.01 mg/kg to about 100 mg/kg bodyweight. In some preferred embodiments the dosage is about 0.1-50 mg/kg;in some preferred embodiments the dosage is about 0.5-10 mg/kg. Thedosage for local administration is typically lower than for systemicadministration. In some embodiments a single administration issufficient; in some embodiments the TWEAKR antagonist is administered asmultiple doses over one or more days.

The TWEAKR antagonists are typically administered in the form of apharmaceutical composition comprising one or more pharmacologicallyacceptable carriers. Pharmaceutically acceptable carriers includediluents, fillers, adjuvants, excipients, and vehicles which arepharmaceutically acceptable for the route of administration, and may beaqueous or oleaginous suspensions formulated using suitable dispersing,wetting, and suspending agents.

Pharmaceutically acceptable carriers are generally sterile and free ofpyrogenic agents, and may include water, oils, solvents, salts, sugarsand other carbohydrates, emulsifying agents, buffering agents,antimicrobial agents, and chelating agents. The particularpharmaceutically acceptable carrier and the ratio of active compound tocarrier are determined by the solubility and chemical properties of thecomposition, the mode of administration, and standard pharmaceuticalpractice.

The compositions as described herein may be contained in a vial, bottle,tube, syringe inhaler or other container for single or multipleadministrations. Such containers may be made of glass or a polymermaterial such as polypropylene, polyethylene, or polyvinylchloride, forexample. Preferred containers may include a seal, or other closuresystem, such as a rubber stopper that may be penetrated by a needle inorder to withdraw a single dose and then re-seal upon removal of theneedle. All such containers for injectable liquids, lyophilizedformulations, reconstituted lyophilized formulations or reconstitutablepowders for injection known in the art or for the administration ofaerosolized compositions are contemplated for use in the presentlydisclosed compositions and methods.

The TWEAKR antagonists are administered to the patient in a mannerappropriate to the indication. Thus, for example, a TWEAKR antagonist,or a pharmaceutical composition thereof, may be administered byintravenous, transdermal, intradermal, intraperitoneal, intramuscular,intranasal, epidural, oral, topical, subcutaneous, intracavity,sustained release from implants, peristaltic routes, or by any othersuitable technique. Parenteral administration is preferred.

In certain embodiments of the claimed invention, the treatment furthercomprises treating the mammal with one or more additionalchemotherapeutic agents. The additional chemotherapeutic agent(s) may beadministered prior to, concurrently with, or following theadministration of the TWEAKR antagonist. The use of more than onechemotherapeutic agent is particularly advantageous when the mammal thatis being treated has a solid tumor. In some embodiments of the claimedinvention, the treatment further comprises treating the mammal withradiation. Radiation, including brachytherapy and teletherapy, may beadministered prior to, concurrently with, or following theadministration of the second chemotherapeutic agent(s) and/or TWEAKRantagonist.

When the mammal that is being treated has a solid tumor, the methodpreferably includes the administration of, in addition to a TWEAKRantagonist, one or more chemotherapeutic agents selected from the groupconsisting of alkylating agents, antimetabolites, vinca alkaloids andother plant-derived chemotherapeutics, nitrosoureas, antitumorantibiotics, antitumor enzymes, topoisomerase inhibitors, platinumanalogs, adrenocortical suppressants, hormones, hormone agonists andantagonists, antibodies, immunotherapeutics, blood cell factors,radiotherapeutics, and biological response modifiers.

In some preferred embodiments the method includes administration of, inaddition to a TWEAKR antagonist, one or more chemotherapeutic agentsselected from the group consisting of cisplatin, cyclophosphamide,mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil,5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine,and vinblastine, lymphokines and cytokines such as interleukins,interferons (including alpha, beta, or delta), and TNF, chlorambucil,busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine,cytarabine, mercaptopurine, thioguanine, vindesine, etoposide,teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine,mitotane, tamoxifen, and fluoxymesterone.

In some preferred embodiments the method includes administration of, inaddition to a TWEAKR antagonist, one or more chemotherapeutic agents,including various soluble forms thereof, selected from the groupconsisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12,4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptorantagonists, TRAIL, VEGF antagonists, VEGF receptor (including VEGF-R1and VEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists, Tekantagonists, and CD148 (also referred to as DEP-1, ECRTP, and PTPRJ, seeTakahashi et al., J. Am. Soc. Nephrol. 10:2135-45, 1999) agonists. Insome preferred embodiments the TWEAKR antagonists of the invention areused as a component of, or in combination with, “metronomic therapy,”such as that described by Browder et al. and Klement et al. (CancerResearch 60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see alsoBarinaga, Science 288:245, 2000).

The polypeptides, compositions, and methods of the present invention maybe used as a first line treatment, for the treatment of residual diseasefollowing primary therapy, or as an adjunct to other therapies includingchemotherapy, surgery, radiation, and other therapeutic methods known inthe art.

When the nucleic acid sequences of the present invention are deliveredaccording to the methods disclosed herein, it is advantageous to use adelivery mechanism so that the sequences will be incorporated into acell for expression. Delivery systems that may advantageously beemployed in the contemplated methods include the use of, for example,viral delivery systems such as retroviral and adenoviral vectors, aswell as non-viral delivery systems. Such delivery systems are well knownby those skilled in the art.

G. Methods of Screening

The TWEAK receptor as described herein may be used in a variety ofmethods of screening to isolate, for example, TWEAKR agonists andantagonists. TWEAKR agonists are compounds that promote the biologicalactivity of TWEAKR and TWEAKR antagonists are compounds that inhibit thebiological activity of TWEAKR. Compounds identified via the followingscreening assays can be used in compositions and methods for modulatingangiogenesis to treat a variety of disease states. The present inventionprovides methods of screening for compounds that (1) modulate TWEAKreceptor or ligand gene expression in a target tissue or cell, (2)modulate the TWEAK receptor-ligand interaction to regulate angiogenesis;(3) bind to the TWEAK receptor or ligand to influence angiogenesis; or(4) interfere with or regulate the bound TWEAK receptor-ligand complex'sinfluence on downstream events such as angiogenesis.

The present invention contemplates the use of assays that are designedto identify compounds that modulate the activity of a TWEAK receptor orligand gene (i.e., modulate the level of TWEAK gene expression and/ormodulate the level of TWEAK gene product activity). Assays mayadditionally be utilized that identify compounds that bind to TWEAK generegulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994,J. Biol. Chem. 269, 28558-28562), and that may modulate the level ofTWEAK gene expression.

Such an assay may involve, for example, the use of a control system, inwhich transcription and translation of the TWEAK receptor or ligand geneoccurs, in comparison to a system including a test compounds suspectedof influencing normal transcription or translation of a TWEAK gene. Forexample, one could determine the rate of TWEAK receptor RNA produced bycardiac cells, and use this to determine if a test compound influencesthat rate. To assess the influence of a test compound suspected toinfluence this normal rate of transcription, one would first determinethe rate of TWEAK receptor RNA production in a cardiac cell culture by,for example, Northern Blotting. One could then administer the testcompound to a cardiac cell culture under otherwise identical conditionsas the control culture. Then the rate of TWEAK receptor RNA in theculture treated with the test compound could be determined by, forexample, Northern Blotting, and compared to the rate of TWEAK receptorRNA produced by the control culture cells. An increase in the TWEAKreceptor RNA in the cells contacted with the test compound relative tocontrol cells is indicative of a stimulator of TWEAK receptor genetranscription and/or translation in cardiac cells, while a decrease isindicative of an inhibitor of TWEAK receptor gene transcription and/ortranslation in cardiac cells.

There are a variety of other methods that can be used to determine thelevel of TWEAK receptor or ligand gene expression as well, and mayfurther be used in assays to determine the influence of a test compoundon the level of TWEAK receptor or ligand gene expression. For example,RNA from a cell type or tissue known, or suspected, to express the TWEAKreceptor or ligand gene, such as cardiac, may be isolated and testedutilizing hybridization or PCR techniques. The isolated cells can bederived from cell culture or from a patient. The analysis of cells takenfrom culture may be a necessary step in the assessment of cells to beused as part of a cell-based gene therapy technique or, alternatively,to test the effect of compounds on the expression of the TWEAK receptoror ligand gene. Such analyses may reveal both quantitative andqualitative aspects of the expression pattern of the TWEAK receptor orligand gene, including activation or inactivation of TWEAK receptor orligand gene expression.

In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). A sequence within the cDNAis then used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the TWEAK receptor or ligand gene nucleicacid segments described above. The preferred lengths of such nucleicacid reagents are at least 9-30 nucleotides. For detection of theamplified product, the nucleic acid amplification may be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product may be made such that the product may bevisualized by standard ethidium bromide staining or by utilizing anyother suitable nucleic acid staining method.

Additionally, it is possible to perform such TWEAK receptor or ligandgene expression assays “in situ”, i.e., directly upon tissue sections(fixed and/or frozen) of patient tissue obtained from biopsies orresections, such that no nucleic acid purification is necessary. TWEAKreceptor or ligand gene nucleic acid segments described above can beused as probes and/or primers for such in situ procedures (see, forexample, Nuovo, G. J., 1992, “PCR In Situ Hybridization: Protocols AndApplications”, Raven Press, New York).

Compounds identified via assays such as those described herein may beuseful, for example, in modulating angiogenesis influenced by the TWEAKreceptor-ligand interaction. Such methods of stimulating or inhibitingTWEAK-influenced angiogenesis are discussed herein.

Alternatively, assay systems may be designed to identify compoundscapable of binding the TWEAK receptor or ligand polypeptide of theinvention and thereby influencing angiogenesis resulting from thisinteraction. Compounds identified may be useful, for example, inmodulating the vascularization of target tissues or cells, may beutilized in screens for identifying compounds that disrupt normal TWEAKreceptor-ligand interactions, or may in themselves disrupt suchinteractions.

The principle of the assays used to identify compounds that bind to theTWEAK receptor or ligand involves preparing a reaction mixture of theTWEAK receptor or ligand and the test compound under conditions and fora time sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay screening for compoundsthat bind to the TWEAK receptor, would involve anchoring the TWEAKreceptor or the test substance onto a solid phase and detecting TWEAKreceptor/test compound complexes anchored on the solid phase at the endof the reaction. In one embodiment of such a method, the TWEAK receptormay be anchored onto a solid surface, and the test compound, which isnot anchored, may be labeled, either directly or indirectly.Alternatively, these same methods could be used to screen for testcompounds that bind to the TWEAK ligand rather than receptor.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored. In order to conduct the assay, the non-immobilizedcomponent is added to the coated surface containing the anchoredcomponent. After the reaction is complete, unreacted components areremoved (e.g., by washing) under conditions such that any complexesformed will remain immobilized on the solid surface. The detection ofcomplexes anchored on the solid surface can be accomplished in a numberof ways. Where the previously non-immobilized component is pre-labeled,the detection of label immobilized on the surface indicates thatcomplexes were formed. Where the previously non-immobilized component isnot pre-labeled, an indirect label can be used to detect complexesanchored on the surface; e.g., using a labeled antibody specific for thepreviously non-immobilized component (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for the TWEAKreceptor or ligand or the test compound to anchor any complexes formedin solution, and a labeled antibody specific for the other component ofthe possible complex to detect anchored complexes.

Those compounds identified as binding agents for either the TWEAKreceptor or the TWEAK ligand may further be assessed for their abilityto interfere with TWEAK receptor-ligand interaction, as described below,and thereby suppress or promote angiogenesis resulting from TWEAKreceptor-ligand interaction. Such compounds may then be usedtherapeutically to stimulate or inhibit angiogenesis.

The TWEAK receptor and ligand polypeptides of the present invention mayalso be used in a screening assay to identify compounds and smallmolecules which specifically interact with the disclosed TWEAK receptoror ligand to either inhibit (antagonize) or enhance (agonize)interaction between these molecules. Thus, for example, polypeptides ofthe invention may be used to identify antagonists and agonists fromcells, cell-free preparations, chemical libraries, and natural productmixtures. The antagonists and agonists may be natural or modifiedsubstrates, ligands, enzymes, receptors, etc. of the polypeptides of theinstant invention, or may be structural or functional mimetics of thepolypeptides. Potential antagonists of the TWEAK receptor-ligandinteraction of the instant invention may include small molecules,peptides, and antibodies that bind to and occupy a binding site of thepolypeptides, causing them to be unavailable to interact and thereforepreventing their normal ability to modulate angiogenesis. Otherpotential antagonists are antisense molecules which may hybridize tomRNA in vivo and block translation of the mRNA into the polypeptides ofthe instant invention. Potential agonists include small molecules,peptides and antibodies which bind to the instant TWEAK polypeptides andinfluence angiogenesis as caused by the disclosed interactions of theTWEAK polypeptides of the instant invention.

Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physiochemical andpharmacological properties that enhance cell penetration, resistdegradation and prolong their physiological half-lives. (Gibbs,“Pharmaceutical Research in Molecular Oncology,” Cell, Vol. 79, (1994).)Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, may be used to bind to and inhibit thepolypeptides of the instant invention by blocking the commencement of asignaling cascade. It is preferable that the antibodies are humanized,and more preferable that the antibodies are human. The antibodies of thepresent invention may be prepared by any of a variety of well-knownmethods.

Specific screening methods are known in the art and many are extensivelyincorporated in high throughput test systems so that large numbers oftest compounds can be screened within a short amount of time. The assayscan be performed in a variety of formats, including protein-proteinbinding assays, biochemical screening assays, immunoassays, cell basedassays, etc. These assay formats are well known in the art. Thescreening assays of the present invention are amenable to screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides and other antagonists andagonists.

One embodiment of a method for identifying molecules which antagonize orinhibit TWEAK receptor-ligand interaction involves adding a candidatemolecule to a medium which contains cells that express the polypeptidesof the instant invention; changing the conditions of said medium sothat, but for the presence of the candidate molecule, the polypeptideswould interact; and observing the binding and inhibition ofangiogenesis. Binding of the TWEAK receptor and ligand can be determinedaccording to competitive binding assays outlined above, and well knownin the art. The angiogenic effect of this binding can be determined viacell proliferation assays such as, for example, cell density assays, orother cell proliferation assays that are also well-known in the art. Theactivity of the cells contacted with the candidate molecule may then becompared with the identical cells which were not contacted and agonistsand antagonists of the TWEAK polypeptide interactions of the instantinvention may be identified. The measurement of biological activity maybe performed by a number of well-known methods such as measuring theamount of protein present (e.g. an ELISA) or of the protein's activity.A decrease in biological stimulation or activation would indicate anantagonist. An increase would indicate an agonist.

Screening assays can further be designed to find molecules that mimicthe biological activity resulting from the TWEAK polypeptideinteractions of the instant invention. Molecules which mimic thebiological activity of a polypeptide may be useful for enhancing thebiological activity of the polypeptide. To identify compounds fortherapeutically active agents that mimic the biological activity of apolypeptide, it must first be determined whether a candidate moleculebinds to the polypeptide. A binding candidate molecule is then added toa biological assay to determine its biological effects. The biologicaleffects of the candidate molecule are then compared to those of thepolypeptide.

Additionally, complex formation within reaction mixtures containing thetest compound and normal TWEAK receptor or ligand gene protein may alsobe compared to complex formation within reaction mixtures containing thetest compound and a mutant TWEAK receptor or ligand gene protein. Thiscomparison may be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normalTWEAK receptor or ligand gene proteins.

The assay for compounds that interfere with the interaction of the TWEAKreceptor or ligand gene products and binding partners can be conductedin a heterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the TWEAK receptor or ligand gene product or thebinding partner onto a solid phase and detecting complexes anchored onthe solid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the TWEAK receptoror ligand gene products and the binding partners, e.g., by competition,can be identified by conducting the reaction in the presence of the testsubstance; i.e., by adding the test substance to the reaction mixtureprior to or simultaneously with the TWEAK receptor and ligand geneproducts. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are described briefly below.

In a heterogeneous assay system, either the TWEAK receptor or ligandgene product, is anchored onto a solid surface, while the non-anchoredspecies is labeled, either directly or indirectly. In practice,microtiter plates are conveniently utilized. The anchored species may beimmobilized by non-covalent or covalent attachments. Non-covalentattachment may be accomplished simply by coating the solid surface witha solution of the TWEAK receptor or ligand gene product and drying.Alternatively, an immobilized antibody specific for the species to beanchored may be used to anchor the species to the solid surface. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds that inhibit complex formation or that disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the TWEAK receptor orligand gene product is prepared in which either the TWEAK receptor orligand gene product or its binding partners is labeled, but the signalgenerated by the label is quenched due to complex formation (see, e.g.,U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt TWEAK receptor or ligand gene productinteraction can be identified.

In a particular embodiment, the TWEAK receptor or ligand gene productcan be prepared for immobilization using recombinant DNA techniques. Forexample, the TWEAK receptor or ligand coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive binding partner can bepurified and used to raise a monoclonal antibody, using methodsroutinely practiced in the art. This antibody can be labeled with theradioactive isotope <125>I, for example, by methods routinely practicedin the art. In a heterogeneous assay, e.g., the GST-TWEAK receptor orligand fusion protein can be anchored to glutathione-agarose beads. TheTWEAK receptor or ligand gene product can then be added in the presenceor absence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed components. Theinteraction between the TWEAK receptor and ligand gene products can bedetected by measuring the amount of radioactivity that remainsassociated with the glutathione-agarose beads. A successful inhibitionof the interaction by the test compound will result in a decrease inmeasured radioactivity.

Alternatively, a GST-TWEAK receptor gene fusion protein and TWEAK ligandgene product (or vice versa) can be mixed together in liquid in theabsence of the solid glutathione-agarose beads. The test compound can beadded either during or after the species are allowed to interact. Thismixture can then be added to the glutathione-agarose beads and unboundmaterial is washed away. Again the extent of inhibition of the TWEAKreceptor-ligand gene product interaction can be detected by adding thelabeled antibody and measuring the radioactivity associated with thebeads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the TWEAK receptor and/or ligand protein, in place of one or both ofthe full length proteins. Any number of methods routinely practiced inthe art can be used to identify and isolate the binding sites. Thesemethods include, but are not limited to, mutagenesis of the geneencoding one of the proteins and screening for disruption of binding ina co-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described in this Sectionabove, and allowed to interact with and bind to its labeled bindingpartner, which has been treated with a proteolytic enzyme, such astrypsin. After washing, a short, labeled peptide comprising the bindingdomain may remain associated with the solid material, which can beisolated and identified by amino acid sequencing. Also, once the genecoding for the segments can be engineered to express peptide fragmentsof the protein, which can then be tested for binding activity andpurified or synthesized.

For example, and not by way of limitation, a TWEAK receptor or ligandgene product can be anchored to a solid material as described, above, inthis Section by making a GST-TWEAK receptor or ligand fusion protein andallowing it to bind to glutathione agarose beads. The interactivebinding partner obtained can be labeled with a radioactive isotope, suchas <35>S, and cleaved with a proteolytic enzyme such as trypsin.Cleavage products can then be added to the anchored GST-TWEAK receptorfusion protein or TWEAK ligand fusion protein and allowed to bind. Afterwashing away unbound peptides, labeled bound material, representing thebinding partner binding domain, can be eluted, purified, and analyzedfor amino acid sequence by well-known methods. Peptides so identifiedcan be produced synthetically or fused to appropriate facilitativeproteins using recombinant DNA technology.

The TWEAK receptor-ligand interactions of the invention, in vivo,initiate a cascade of events that either stimulate or suppressangiogenesis in a target group of cell or tissue. Molecules, such asnucleic acid molecules, proteins, or small molecules may, in turn,influence this cascade. Compounds that disrupt the TWEAK receptor-ligandinteraction effects in this way may be useful in regulatingangiogenesis.

The basic principle of the assay systems used to identify compounds thatinterfere with the angiogenic or anti-angiogenic effect of TWEAKreceptor-ligand interaction involves preparing a reaction mixturecontaining the TWEAK receptor and ligand under conditions and for a timesufficient to allow the two to interact and bind, thus forming acomplex. In order to test a compound for inhibitory activity of theeffect of this interaction, the reaction mixture is prepared in thepresence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of the TWEAK receptor-ligand complex. Controlreaction mixtures are incubated without the test compound or with aplacebo. The inhibition or potentiation of any effect of the TWEAKcomplex on vascularization is then detected. Normal angiogenic responsein the control reaction, but not in the reaction mixture containing thetest compound, indicates that the compound interferes with the cascadeof events initiated by the TWEAK receptor-ligand interaction. Enhancedangiogenesis in the test compounds-containing culture indicates astimulator of the TWEAK receptor-ligand complex effect.

EXAMPLES

The following examples are intended to illustrate particular embodimentsand not to limit the scope of the invention.

Example 1 Identification of the TWEAK Receptor

A. Expression Cloning of TWEAK Receptor cDNA

To clone TWEAK Receptor cDNA, an expression vector encoding a growthhormone leader, a leucine zipper multimerization domain, and theC-terminal extracellular domain of human TWEAK (see Chicheportiche etal., J. Biol. Chem. 272(51):32401, 1997) was constructed. Thisexpression vector, which was named pDC409-LZ-TWEAK, comprised the DNAsequence SEQ ID NO:1 and encoded the polypeptide SEQ ID NO:2.pDC409-LZ-TWEAK conditioned supernatants were produced by transienttransfection into CV1-EBNA cells. These supernatants were incubated withmagnetic beads coated with polyclonal goat anti-mouse antibody that hadpreviously been incubated with a mouse monoclonal antibody against theleucine zipper. Control beads were produced by mixing the coated beadswith supernatants from cells transfected with empty vector.

A monolayer of COS cells grown in a T175 flask was transfected with 15μg of DNA pools of complexity of 100,000 from a HUVEC cDNA expressionlibrary. After 2 days these cells were lifted from the flask, andincubated in 1.5 mls of binding media plus 5% non-fat dried milk for 3hours at 4 degrees C. on a rotator wheel. Cells were pre-cleared byadding control beads and rotated at 4 degrees C. for an additional 45minutes after which bead bound cells were removed with a magnet.Pre-clearing was repeated 2-3 times, then TWEAK coated beads were addedto the cells and rotated 30 minutes at 4 degrees C. Cells binding theTWEAK beads were separated by use of a magnet and washed 4× in PBS.Plasmid DNA was extracted from these cells by lysing in 0.1% SDS, andelectroporating the supernatants in DH101B cells. Colonies were grownovernight on ampicilin selective media. Transformants were pooled andused as a source of plasmid DNA for a further round of panning. After 2rounds of panning, positive clones were picked from the resulting poolbased on their ability to bind TWEAK using a slide binding protocol likethat described in Part B, below.

The human TWEAK receptor (also called TWEAKR) cDNA was determined tohave the sequence SEQ ID NO:3, which encodes a 129 residue polypeptide(SEQ ID NO:4). Examination of the sequence predicts a polypeptide havingan approximately 78 amino acid extracellular domain (residues 1-78 ofSEQ ID NO:4, including the signal peptide), an approximately 23 aminoacid transmembrane domain (residues 79-101 of SEQ ID NO:4), and anapproximately 28 amino acid intracellular domain (residues 102-129 ofSEQ ID NO:4). TWEAKR is the smallest known TNF receptor family member.It has a single cysteine-rich repeat region in the extracellular domain,as compared to the 3-4 repeats of other TNF receptor family members. TheTWEAKR polypeptide was previously described as a transmembrane proteinencoded by a human liver cDNA clone (WO 98/55508, see also WO 99/61471),but had not been identified as the TWEAK receptor. A murine homolog, theFGF-inducible Fn14 (Meighan-Mantha et al., J. Biol. Chem. 274(46):33166,1999), is approximately 82% identical to the human protein, as shown bythe alignment in FIG. 1.

The newly identified TWEAK receptor was tested side by side with DR3(which had been identified as the TWEAK receptor by Marsters et al.,Current Biology 8:525, 1998) for the ability to bind to TWEAK.

B. The TWEAK Receptor Binds to TWEAK

Slides of COS cells were transfected with expression vectors containingTWEAKR, DR3, or vector without insert (control). After two days thecells were incubated with concentrated supernatants from CV-1 cellstransfected with a vector encoding the leucine zipper TWEAKextracellular domain fusion protein. One hour later the cells werewashed and probed with an I-125 labeled antibody against theleucine-zipper domain. The slides were washed, fixed, andautoradiography was performed using x-ray film. The TWEAKR transfectedcells bound significant amounts of TWEAK. TWEAK did not bind to thecells transfected with DR3 or the control cells. This experimentconfirmed that the TWEAKR polypeptide identified in part A above, ratherthan DR3, is the major receptor for TWEAK. After discovery of thefunctional TWEAK receptor, other investigators also reported that DR3 isnot the major receptor for TWEAK (Kaptein et al., FEBS Lett.,485(2-3):135, 2000. The TWEAK-TWEAKR binding interaction was furthercharacterized by Scatchard analysis.

CV-1 cells were transfected with human full length TWEAK and mixed 1:30with Raji cells, which do not express TWEAK. The cells were incubatedwith serial dilutions of 125-I labeled human TWEAK receptor-Fc for 2hours at 4 degrees Celsius. Free and bound probe was separated bymicrofuging the samples through a phalate oil mixture in plastic tubes.Supernatants and pellets were gamma-counted. Scatchard analyses of TWEAKligand binding the TWEAK receptor showed a binding affinity constant(Ka) of approximately 4.5×10⁸ M⁻¹.

C. The TWEAK Receptor is Strongly Expressed in Cardiac Tissue

To determine the expression pattern of the TWEAK receptor, Northern blotanalyses were performed. Human multiple tissue northern blots werepurchased from Clontech (Palo Alto, Calif.) and probed with P-32 labeledrandom primed DNA from the TWEAK receptor coding region. The blots werewashed and autoradiography was performed using x-ray film. Resultsshowed that in the adult TWEAKR is strongly expressed in heart,placenta, and some skeletal muscle samples. Strong expression in hearttissue further supports the utility of TWEAKR in the diagnosis andtreatment of cardiac disease. In contrast to the adult, the fetaltissues expressed TWEAKR more ubiquitously; TWEAKR transcripts were seenin the lung and liver.

Example 2 Preparation of TWEAKR Antagonists and Agonists

Because TWEAK induces angiogenesis, TWEAKR agonists (such as agonisticantibodies) may be used to promote angiogenesis and TWEAKR antagonists(such as soluble receptors and antagonistic antibodies) may be used toinhibit angiogenesis.

A. Recombinant Production of Soluble TWEAK Receptor-Fc (TWEAKR-Fc)Fusion Polypeptides

To construct a nucleic acid encoding the TWEAKR extracellular domainfused to Fc, a nucleic acid encoding the N-terminal 79 amino acids fromTWEAKR, including the leader (signal peptide), was joined to a nucleicacid encoding an Fc portion from human IgG1. Sequences for thisconstruct are shown as SEQ ID NO:6 (nucleic acid) and SEQ ID NO:7 (aminoacid). In SEQ ID NO:7, residues 1-27 are the predicted signal peptide(predicted to be cleaved upon secretion from the cell; the actualcleavage site was identified by N-terminal sequence analysis, seebelow), residues 28-79 are from the cysteine-rich TWEAKR extracellulardomain, residues 80-81 are from a BglII cloning site, and the remainderis the Fc portion. Upon insertion into a mammalian expression vector,and expression in and secretion from a mammalian host cells, thisconstruct produced a polypeptide designated TWEAKR-Fc. N-terminalsequence analysis determined that the secreted polypeptide designatedTWEAKR-Fc had an N-terminus corresponding to residue 28 (Glu) of SEQ IDNO:7. Anti-angiogenic activity of TWEAKR-Fc was demonstrated usingassays such as those described in the following examples. An analogousFc-fusion construct was prepared using the murine TWEAKR extracellulardomain.

B. Production of Antibodies that Bind the TWEAKR Extracellular Domain

BALB/c mice are immunized with TWEAKR extracellular domain and spleencells are collected and used to prepare hybridomas using standardprocedures. Hybridoma supernatants are screened, using ELISA, for theability to bind TWEAKR. Positives are cloned two times, to insuremonoclonality, then isotyped and reassayed for reactivity to TWEAKR.Antibodies and antibody derivatives are also prepared using transgenicmice that express human immunoglobulins and through the use of phagedisplay. The resulting antibodies are tested in assays such as thosedescribed in the examples below, to characterize their ability tomodulate the TWEAK-TWEAKR interaction, TWEAKR signaling, angiogenesis,and other downstream biological activities.

Agonistic antibodies are used to promote TWEAK-induced biologicalactivities such as angiogenesis, and antagonistic antibodies are used toinhibit TWEAK-induced biological activities such as angiogenesis. Forsome applications, the activity of antagonistic antibodies is augmentedby conjugation to a radioisotope, to a plant-, fungus-, orbacterial-derived cytotoxin such as ricin A or diptheria toxin, or toanother chemical poison. And because of the restricted tissuedistribution of TWEAKR, antibodies that bind to TWEAKR are particularlyuseful as targeting agents for imaging or delivering therapeutics to thevasculature. Antibodies that bind TWEAKR can be used, for example, totarget a detectable label or chemotherapeutic to the mural cells(pericytes and vascular smooth muscle cells). Detectable labels mayinclude radioisotopes, chemiluminescent and fluorescent compounds, andenzymes. These techniques are useful, for example, in the diagnosis,staging, and treatment of neoplasms.

Example 3 Activity of TWEAKR-Fc in a Wound Closure Assay

A planar endothelial cell migration (wound closure) assay was used toquantitate the inhibition of angiogenesis by TWEAKR-Fc in vitro. In thisassay, endothelial cell migration is measured as the rate of closure ofa circular wound in a cultured cell monolayer. The rate of wound closureis linear, and is dynamically regulated by agents that stimulate andinhibit angiogenesis in vivo.

Primary human renal microvascular endothelial cells, HRMEC, wereisolated, cultured, and used at the third passage after thawing, asdescribed in Martin et al., In Vitro Cell Dev Biol 33:261, 1997.Replicate circular lesions, “wounds,” (600-800 micron diameter) weregenerated in confluent HRMEC monolayers using a silicon-tipped drillpress. At the time of wounding the medium (DMEM+1% BSA) was supplementedwith 20 ng/ml PMA (phorbol-12-myristate-13-acetate), EGF (4 ng/ml), and0.150 to 5 μg/ml TWEAKR-Fc, or a combination of 40 ng/ml EGF and 0.150to 5 μg/ml TWEAKR-Fc. The residual wound area was measured as a functionof time (0-12 hours) using a microscope and image analysis software(Bioquant, Nashville, Tenn.). The relative migration rate was calculatedfor each agent and combination of agents by linear regression ofresidual wound area plotted over time. The results are shown in FIGS.2-3.

Compared to huIgG or media+BSA, TWEAKR-Fc inhibited PMA-inducedendothelial migration in a dose responsive manner, reducing the rate ofmigration to unstimulated levels at 5 μg/ml (FIG. 2). Neither huIgG norTWEAKR-Fc inhibited basal (uninduced) migration. When HRMEC migrationwas induced by EGF, TWEAKR-Fc inhibited endothelial migration in adose-dependent manner, reducing the rate of migration to unstimulatedlevels at 5 μg/ml (FIG. 3).

Example 4 Activity of TWEAKR-Fc in a Corneal Pocket Assay

A mouse corneal pocket assay was used to quantitate the inhibition ofangiogenesis by TWEAKR-Fc in vivo. In this assay, agents to be testedfor angiogenic or anti-angiogenic activity are immobilized in a slowrelease form in a hydron pellet, which is implanted into micropocketscreated in the corneal epithelium of anesthetized mice. Vascularizationis measured as the appearance, density, and extent of vessel ingrowthfrom the vascularized corneal limbus into the normally avascular cornea.

Hydron pellets, as described in Kenyon et al., Invest Opthamol. & VisualScience 37:1625, 1996, incorporated sucralfate with bFGF (90 ng/pellet),bFGF and IgG (14 μg/pellet, control), or bFGF and TWEAKR-Fc (14 μg). Thepellets were surgically implanted into corneal stromal micropocketscreated by micro-dissection 1 mm medial to the lateral corneal limbus of6-8 week old male C57BL mice. After five days, at the peak ofneovascular response to bFGF, the corneas were photographed, using aZeiss slit lamp, at an incipient angle of 35-50° from the polar axis inthe meridian containing the pellet. Images were digitized and processedby subtractive color filters (Adobe Photoshop 4.0) to delineateestablished microvessels by hemoglobin content. Image analysis software(Bioquant, Nashville, Tenn.) was used to calculate the fraction of thecorneal image that was vascularized, the vessel density within thevascularized area, and the vessel density within the total cornea.

As shown in Table 1, TWEAKR-Fc (100 pmol) inhibited bFGF (3pmol)-induced corneal angiogenesis, reducing the vascular density to 50%of that induced by FGF alone or FGF+IgG.

TABLE 1 Effect of TWEAKR-Fc on FGF-induced Angiogenesis in the MouseCorneal Pocket Assay Greater than 50% Reduction in Number and Length ofVessels Treatment n/total n (%) FGF alone 0/2 (0%) FGF + IgG 0/2 (0%)FGF + TWEAKR-Fc 6/9 (67%) 

Example 5 Qualitative TRAF Binding to the TWEAK Receptor (TWEAKR)Cytoplasmic Domain

Members of the TRAF family are intra-cellular signaling molecules.Several members of the TRAF family are known to associate with membersof the TNF receptor family in order to initiate a signaling cascade thatactivates the NF-kappa-B pathway, resulting in cell activation andproliferation. A qualitative in vitro binding assay was performed totest whether members of the TRAF family of intra-cellular signalingmolecules bind to the cytoplasmic domain of TWEAKR and to learn,therefore, whether the small cytoplasmic domain of TWEAKR is capable ofmediating a signal into the cell via the TRAF pathway, A GST fusionvector consisting of the C-terminal 29 amino acids of TWEAKR fused toglutathione S-transferase was created by sub-cloning the appropriateinsert into the pGEX-4T (Amersham Pharmacia Biotech) vector at the BamHIand NotI sites. The product from this vector was expressed in E. coliand bound to sepharose beads as described by Galibert et al., J. Biol.Chem. 273(51):34120, 1998. Similarly constructed beads coated with RANKcytoplasmic domain-GST fusion proteins were used as a positive control,and beads coated with GST alone were used as a negative control.[35S]methionine/cysteine labeled TRAF proteins were produced inreticulocyte lysates (TNT-coupled Reticulocyte Lysate Systems, Promega)according to the manufacturer's protocol. Reticulocyte lysatescontaining the labeled TRAF molecules were first pre-cleared using thecontrol beads followed incubation with the indicated fusion proteincoated beads in binding buffer (50 mM HEPES [pH 7.4], 250 mM NaCl, 0.25%(v/v) Nonidet P-40, 10% glycerol, 2 mM EDTA) at 4 degrees Celsius for 2hours. After washing 4×with binding buffer bound TRAF molecules elutedfrom the beads in SDS-loading buffer, separated by SDS-PAGE, dried andexposed to X-ray film.

Binding above background levels was seen with TRAFS 1,2 and 3. Nobinding above background levels was seen with TRAFS 4,5, and 6. Theability of TWEAKR to bind to TRAFs 1,2, and 3 demonstrates that TWEAKRis capable of inducing a signal to the cell via the TRAF pathway, andtherefore transmitting a proliferative signal into the host cell. Thisexperiment provides further evidence that TWEAKR is the functionalreceptor for TWEAK. It also illustrates a further means by whichsignaling can be inhibited: by disrupting the TRAF-TWEAKR interactionwith a small molecule, or by use of a dominant negative variant of theTRAF molecule.

Example 6 Activity of TWEAKR-Fc in an Endothelial Cell ProliferationAssay

An endothelial cell proliferation assay was used to quantitate theinhibition of bFGF or TWEAK induced-proliferation by TWEAKR-Fc in vitro.In this assay, endothelial cell proliferation is measured after 4 daysof cell growth in microtiter wells using a cell labeling molecule calledcalcein AM. Esterases expressed by the cells cleave the calcein andcause it to fluoresce when excited at 485 nm. Uncleaved calcein does notfluoresce. The amount of fluorescence is directly related to the numberof endothelial cells in the culture well. Endothelial cell proliferationis often regulated by agents that stimulate and/or inhibit angiogenesisin vivo.

Primary HUVEC (human umbilical vein endothelial cells) were obtainedfrom a commercial source (Clonetics, Walkersville, Md.), cultured, andused at passage 2 to 7. Replicate cultures were set up by adding 3000HUVEC to each microtiter well in endothelial cell basal media (EBM, anendothelial cell basal media that contains no growth factors or serumand is based on the media formulations developed by Dr. Richard Ham atthe University of Colorado, Clonetics) plus 0.05% FBS (fetal bovineserum). At the time of culture initiation FGF-2 (fibroblast growthfactor-2, 10 ng/ml) or human TWEAK (100 ng/ml) was added to the culturesin the presence of human IgG (huIgG, control) or human TWEAKR-Fc atconcentrations ranging from 0.08 μg/ml to 20 μg/ml (0.25 to 20 μg/ml forTWEAK-induced and 0.08 to 6.7 μg/ml for FGF 2-induced). The HUVECcontaining cultures were incubated for 4 days at 37 degrees C., 5% C0₂.On the fourth day of culture 4 μM calcein-AM was added to the culturesand 2 hours later the wells were evaluated for fluorescence. Theresults, expressed as the average fluorescence (485-530 nm) counts forreplicate wells plus or minus the SEM, are shown in FIGS. 4 and 5.

TWEAKR-Fc specifically inhibited TWEAK-induced HUVEC proliferation in adose-dependent manner when compared to huIgG which did not effectTWEAK-induced proliferation (FIG. 4). In addition, TWEAKR-Fc inhibitedthe basal proliferation of HUVEC observed during culture in EBM plus0.05% FBS, as compared to huIgG which did not. Interestingly, TWEAKR-Fcalso inhibited FGF-2 mediated HUVEC proliferation at concentrations ofgreater than 2 μg/ml, as compared to huIgG which did not effect theFGF-2 induced HUVEC proliferative response (FIG. 5). These results showthat TWEAKR-Fc inhibits HUVEC proliferation induced by the addition ofexogenous recombinant human TWEAK. That TWEAKR-Fc partially inhibitsserum -induced HUVEC-proliferation indicates HUVEC produce endogenousTWEAK that promotes growth/survival of the EC (endothelial cell) via theTWEAKR.

TWEAKR-Fc attenuation of FGF-2 induced proliferation indicates that atleast part of the EC response to FGF-2 is dependent on endogenousTWEAK/TWEAKR interaction.

Example 7 Inhibition of Neovascularization by TWEAKR Antagonists in aMurine Cardiac Ischemia/Engraftment Model

Survival of heterotopically transplanted cardiac tissue from one mousedonor to the ear skin of another genetically similar mouse requiresadequate neovascularization by the transplanted heart and thesurrounding tissue, to promote survival and energy for cardiac musclefunction. Inadequate vasculature at the site of transplant causesexcessive ischemia to the heart, tissue damage, and failure of thetissue to engraft. Agents that antagonize factors involved inendothelial cell migration and vessel formation can decreaseangiogenesis at the site of transplant, thereby limiting graft tissuefunction and ultimately engraftment itself. A murine heterotopic cardiacisograft model is used to demonstrate the effects of TWEAKR antagonists,including antibodies and TWEAKR-Fc, on neovascularization.

Female BALB/c (≈12 weeks of age) recipients are given neonatal heartgrafts from donor mice of the same strain. The donor heart tissue isgrafted into the left ear pinnae of the recipient on day 0 and the miceare divided into two groups. The control group receives human IgG (HuIgG) while the other group receives the TWEAKR antagonist, bothintraperitoneally. The treatments are continued for five consecutivedays. The functionality of the grafts is determined by monitoringvisible pulsatile activity on days 7 and 14 post-engraftment. Theinhibition of functional engraftment, as a function of the dose ofTWEAKR antagonist, is determined. The histology of the transplantedhearts is examined is order to visualize the effects of the TWEAKRantagonist on edema at the site of transplant and host and donor tissuevasculature (using, e.g., Factor VIII staining).

Example 6 Treatment of Tumors with TWEAKR Antagonists

TWEAKR antagonists, including antibodies and TWEAKR-Fc, are tested inanimal models of solid tumors. The effect of the TWEAKR antagonists isdetermined by measuring tumor frequency and tumor growth.

The relevant disclosures of publications cited herein are specificallyincorporated by reference. The examples presented above are not intendedto be exhaustive or to limit the scope of the invention. The skilledartisan will understand that variations and modifications and variationsare possible in light of the above teachings, and such modifications andvariations are intended to be within the scope of the invention.

7 1 898 DNA Artificial Sequence CDS (52)..(873) Description ofArtificial Sequence human TWEAK fusion protein construct 1 tctcgagggccacgcgttta aacgtcgagg tacctatccc gggccgccac c atg gct 57 Met Ala 1 acaggc tcc cgg acg tcc ctg ctc ctg gct ttt ggc ctg ctc tgc ctg 105 Thr GlySer Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu 5 10 15 ccc tggctt caa gag ggc agt gca act agt tct gac cgt atg aaa cag 153 Pro Trp LeuGln Glu Gly Ser Ala Thr Ser Ser Asp Arg Met Lys Gln 20 25 30 ata gag gataag atc gaa gag atc cta agt aag att tat cat ata gag 201 Ile Glu Asp LysIle Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu 35 40 45 50 aat gaa atcgcc cgt atc aaa aag ctg att ggc gag cgg act aga tct 249 Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly Glu Arg Thr Arg Ser 55 60 65 agt ttg ggg agccgg gca tcg ctg tcc gcc cag gag cct gcc cag gag 297 Ser Leu Gly Ser ArgAla Ser Leu Ser Ala Gln Glu Pro Ala Gln Glu 70 75 80 gag ctg gtg gca gaggag gac cag gac ccg tcg gaa ctg aat ccc cag 345 Glu Leu Val Ala Glu GluAsp Gln Asp Pro Ser Glu Leu Asn Pro Gln 85 90 95 aca gaa gaa agc cag gatcct gcg cct ttc ctg aac cga cta gtt cgg 393 Thr Glu Glu Ser Gln Asp ProAla Pro Phe Leu Asn Arg Leu Val Arg 100 105 110 cct cgc aga agt gca cctaaa ggc cgg aaa aca cgg gct cga aga gcg 441 Pro Arg Arg Ser Ala Pro LysGly Arg Lys Thr Arg Ala Arg Arg Ala 115 120 125 130 atc gca gcc cat tatgaa gtt cat cca cga cct gga cag gac gga gcg 489 Ile Ala Ala His Tyr GluVal His Pro Arg Pro Gly Gln Asp Gly Ala 135 140 145 cag gca ggt gtg gacggg aca gtg agt ggc tgg gag gaa gcc aga atc 537 Gln Ala Gly Val Asp GlyThr Val Ser Gly Trp Glu Glu Ala Arg Ile 150 155 160 aac agc tcc agc cctctg cgc tac aac cgc cag atc ggg gag ttt ata 585 Asn Ser Ser Ser Pro LeuArg Tyr Asn Arg Gln Ile Gly Glu Phe Ile 165 170 175 gtc acc cgg gct gggctc tac tac ctg tac tgt cag gtg cac ttt gat 633 Val Thr Arg Ala Gly LeuTyr Tyr Leu Tyr Cys Gln Val His Phe Asp 180 185 190 gag ggg aag gct gtctac ctg aag ctg gac ttg ctg gtg gat ggt gtg 681 Glu Gly Lys Ala Val TyrLeu Lys Leu Asp Leu Leu Val Asp Gly Val 195 200 205 210 ctg gcc ctg cgctgc ctg gag gaa ttc tca gcc act gcg gcc agt tcc 729 Leu Ala Leu Arg CysLeu Glu Glu Phe Ser Ala Thr Ala Ala Ser Ser 215 220 225 ctc ggg ccc cagctc cgc ctc tgc cag gtg tct ggg ctg ttg gcc ctg 777 Leu Gly Pro Gln LeuArg Leu Cys Gln Val Ser Gly Leu Leu Ala Leu 230 235 240 cgg cca ggg tcctcc ctg cgg atc cgc acc ctc ccc tgg gcc cat ctc 825 Arg Pro Gly Ser SerLeu Arg Ile Arg Thr Leu Pro Trp Ala His Leu 245 250 255 aag gct gcc cccttc ctc acc tac ttc gga ctc ttc cag gtt cac tga 873 Lys Ala Ala Pro PheLeu Thr Tyr Phe Gly Leu Phe Gln Val His 260 265 270 gcggccgcggatctgtttaa actag 898 2 273 PRT Artificial Sequence Description ofArtificial Sequence human TWEAK fusion protein construct 2 Met Ala ThrGly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys LeuPro Trp Leu Gln Glu Gly Ser Ala Thr Ser Ser Asp Arg Met 20 25 30 Lys GlnIle Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His 35 40 45 Ile GluAsn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg Thr 50 55 60 Arg SerSer Leu Gly Ser Arg Ala Ser Leu Ser Ala Gln Glu Pro Ala 65 70 75 80 GlnGlu Glu Leu Val Ala Glu Glu Asp Gln Asp Pro Ser Glu Leu Asn 85 90 95 ProGln Thr Glu Glu Ser Gln Asp Pro Ala Pro Phe Leu Asn Arg Leu 100 105 110Val Arg Pro Arg Arg Ser Ala Pro Lys Gly Arg Lys Thr Arg Ala Arg 115 120125 Arg Ala Ile Ala Ala His Tyr Glu Val His Pro Arg Pro Gly Gln Asp 130135 140 Gly Ala Gln Ala Gly Val Asp Gly Thr Val Ser Gly Trp Glu Glu Ala145 150 155 160 Arg Ile Asn Ser Ser Ser Pro Leu Arg Tyr Asn Arg Gln IleGly Glu 165 170 175 Phe Ile Val Thr Arg Ala Gly Leu Tyr Tyr Leu Tyr CysGln Val His 180 185 190 Phe Asp Glu Gly Lys Ala Val Tyr Leu Lys Leu AspLeu Leu Val Asp 195 200 205 Gly Val Leu Ala Leu Arg Cys Leu Glu Glu PheSer Ala Thr Ala Ala 210 215 220 Ser Ser Leu Gly Pro Gln Leu Arg Leu CysGln Val Ser Gly Leu Leu 225 230 235 240 Ala Leu Arg Pro Gly Ser Ser LeuArg Ile Arg Thr Leu Pro Trp Ala 245 250 255 His Leu Lys Ala Ala Pro PheLeu Thr Tyr Phe Gly Leu Phe Gln Val 260 265 270 His 3 868 DNA Homosapiens CDS (53)..(442) 3 gcttgaattc aataactata acggtcctaa ggtagcgaagaggacgtgca ct atg gct 58 Met Ala 1 cgg ggc tcg ctg cgc cgg ttg ctg cggctc ctc gtg ctg ggg ctc tgg 106 Arg Gly Ser Leu Arg Arg Leu Leu Arg LeuLeu Val Leu Gly Leu Trp 5 10 15 ctg gcg ttg ctg cgc tcc gtg gcc ggg gagcaa gcg cca ggc acc gcc 154 Leu Ala Leu Leu Arg Ser Val Ala Gly Glu GlnAla Pro Gly Thr Ala 20 25 30 ccc tgc tcc cgc ggc agc tcc tgg agc gcg gacctg gac aag tgc atg 202 Pro Cys Ser Arg Gly Ser Ser Trp Ser Ala Asp LeuAsp Lys Cys Met 35 40 45 50 gac tgc gcg tct tgc agg gcg cga ccg cac agcgac ttc tgc ctg ggc 250 Asp Cys Ala Ser Cys Arg Ala Arg Pro His Ser AspPhe Cys Leu Gly 55 60 65 tgc gct gca gca cct cct gcc ccc ttc cgg ctg ctttgg ccc atc ctt 298 Cys Ala Ala Ala Pro Pro Ala Pro Phe Arg Leu Leu TrpPro Ile Leu 70 75 80 ggg ggc gct ctg agc ctg acc ttc gtg ctg ggg ctg ctttct ggc ttt 346 Gly Gly Ala Leu Ser Leu Thr Phe Val Leu Gly Leu Leu SerGly Phe 85 90 95 ttg gtc tgg aga cga tgc cgc agg aga gag aag ttc acc accccc ata 394 Leu Val Trp Arg Arg Cys Arg Arg Arg Glu Lys Phe Thr Thr ProIle 100 105 110 gag gag acc ggc gga gag ggc tgc cca gct gtg gcg ctg atccag tga 442 Glu Glu Thr Gly Gly Glu Gly Cys Pro Ala Val Ala Leu Ile Gln115 120 125 caatgtgccc cctgccagcc ggggctcgcc cactcatcat tcattcatccattctagagc 502 cagtctctgc ctcccagacg cggcgggagc caagctcctc caaccacaaggggggtgggg 562 ggcggtgaat cacctctgag gcctgggccc agggttcagg ggaaccttccaaggtgtctg 622 gttgccctgc ctctggctcc agaacagaaa gggagcctca cgctggctcacacaaaacag 682 ctgacactga ctaaggaact gcagcatttg cacaggggag gggggtgccctccttcctag 742 aggccctggg ggccaggctg acttgggggg cagacttgac actaggccccactcactcag 802 atgtcctgaa attccaccac gggggtcacc ctggggggtt agggacctatttttaacact 862 agaggg 868 4 129 PRT Homo sapiens 4 Met Ala Arg Gly SerLeu Arg Arg Leu Leu Arg Leu Leu Val Leu Gly 1 5 10 15 Leu Trp Leu AlaLeu Leu Arg Ser Val Ala Gly Glu Gln Ala Pro Gly 20 25 30 Thr Ala Pro CysSer Arg Gly Ser Ser Trp Ser Ala Asp Leu Asp Lys 35 40 45 Cys Met Asp CysAla Ser Cys Arg Ala Arg Pro His Ser Asp Phe Cys 50 55 60 Leu Gly Cys AlaAla Ala Pro Pro Ala Pro Phe Arg Leu Leu Trp Pro 65 70 75 80 Ile Leu GlyGly Ala Leu Ser Leu Thr Phe Val Leu Gly Leu Leu Ser 85 90 95 Gly Phe LeuVal Trp Arg Arg Cys Arg Arg Arg Glu Lys Phe Thr Thr 100 105 110 Pro IleGlu Glu Thr Gly Gly Glu Gly Cys Pro Ala Val Ala Leu Ile 115 120 125 Gln5 129 PRT Mus sp. 5 Met Ala Pro Gly Trp Pro Arg Ser Leu Pro Gln Ile LeuVal Leu Gly 1 5 10 15 Phe Gly Leu Val Leu Met Arg Ala Ala Ala Gly GluGln Ala Pro Gly 20 25 30 Thr Ser Pro Cys Ser Ser Gly Ser Ser Trp Ser AlaAsp Leu Asp Lys 35 40 45 Cys Met Asp Cys Ala Ser Cys Pro Ala Arg Pro HisSer Asp Phe Cys 50 55 60 Leu Gly Cys Ala Ala Ala Pro Pro Ala His Phe ArgLeu Leu Trp Pro 65 70 75 80 Ile Leu Gly Gly Ala Leu Ser Leu Val Leu ValLeu Ala Leu Val Ser 85 90 95 Ser Phe Leu Val Trp Arg Arg Cys Arg Arg ArgGlu Lys Phe Thr Thr 100 105 110 Pro Ile Glu Glu Thr Gly Gly Glu Gly CysPro Gly Val Ala Leu Ile 115 120 125 Gln 6 932 DNA Artificial SequenceCDS (1)..(930) Description of Artificial Sequence human TWEAK receptorfusion protein construct 6 atg gct cgg ggc tcg ctg cgc cgg ttg ctg cggctc ctc gtg ctg ggg 48 Met Ala Arg Gly Ser Leu Arg Arg Leu Leu Arg LeuLeu Val Leu Gly 1 5 10 15 ctc tgg ctg gcg ttg ctg cgc tcc gtg gcc ggggag caa gcg cca ggc 96 Leu Trp Leu Ala Leu Leu Arg Ser Val Ala Gly GluGln Ala Pro Gly 20 25 30 acc gcc ccc tgc tcc cgc ggc agc tcc tgg agc gcggac ctg gac aag 144 Thr Ala Pro Cys Ser Arg Gly Ser Ser Trp Ser Ala AspLeu Asp Lys 35 40 45 tgc atg gac tgc gcg tct tgc agg gcg cga ccg cac agcgac ttc tgc 192 Cys Met Asp Cys Ala Ser Cys Arg Ala Arg Pro His Ser AspPhe Cys 50 55 60 ctg ggc tgc gct gca gca cct cct gcc ccc ttc cgg ctg ctttgg aga 240 Leu Gly Cys Ala Ala Ala Pro Pro Ala Pro Phe Arg Leu Leu TrpArg 65 70 75 80 tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cctgaa gcc 288 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluAla 85 90 95 gag ggc gcg ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gacacc 336 Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr100 105 110 ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gacgtg 384 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val115 120 125 agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggcgtg 432 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val130 135 140 gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aacagc 480 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser145 150 155 160 acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gactgg ctg 528 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp TrpLeu 165 170 175 aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctccca gcc 576 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu ProAla 180 185 190 ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cgagaa cca 624 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg GluPro 195 200 205 cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aagaac cag 672 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys AsnGln 210 215 220 gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gacatc gcc 720 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla 225 230 235 240 gtg gag tgg gag agc aat ggg cag ccg gag aac aac tacaag acc acg 768 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr LysThr Thr 245 250 255 cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tatagc aag ctc 816 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu 260 265 270 acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttctca tgc tcc 864 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe SerCys Ser 275 280 285 gtg atg cat gag gct ctg cac aac cac tac acg cag aagagc ctc tcc 912 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu Ser 290 295 300 ctg tct ccg ggt aaa tga ac 932 Leu Ser Pro Gly Lys305 310 7 309 PRT Artificial Sequence Description of Artificial Sequencehuman TWEAK receptor fusion protein construct 7 Met Ala Arg Gly Ser LeuArg Arg Leu Leu Arg Leu Leu Val Leu Gly 1 5 10 15 Leu Trp Leu Ala LeuLeu Arg Ser Val Ala Gly Glu Gln Ala Pro Gly 20 25 30 Thr Ala Pro Cys SerArg Gly Ser Ser Trp Ser Ala Asp Leu Asp Lys 35 40 45 Cys Met Asp Cys AlaSer Cys Arg Ala Arg Pro His Ser Asp Phe Cys 50 55 60 Leu Gly Cys Ala AlaAla Pro Pro Ala Pro Phe Arg Leu Leu Trp Arg 65 70 75 80 Ser Cys Asp LysThr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 85 90 95 Glu Gly Ala ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 100 105 110 Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 115 120 125 Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 130 135 140 GluVal His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 145 150 155160 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 165170 175 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala180 185 190 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg GluPro 195 200 205 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr LysAsn Gln 210 215 220 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro SerAsp Ile Ala 225 230 235 240 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr 245 250 255 Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu 260 265 270 Thr Val Asp Lys Ser Arg Trp Gln GlnGly Asn Val Phe Ser Cys Ser 275 280 285 Val Met His Glu Ala Leu His AsnHis Tyr Thr Gln Lys Ser Leu Ser 290 295 300 Leu Ser Pro Gly Lys 305

I claim:
 1. A method of inhibiting angiogenesis in a mammal in need ofsuch treatment comprising administering a therapeutically-effectiveamount of a composition comprising an antagonist of a TWEAK receptor,wherein the TWEAK receptor comprises a sequence as set forth from aminoacids 28-79 of SEQ ID No: 7, and the antagonist is selected from thegroup consisting of a soluble TWEAK receptor that comprises thecysteine-rich repeat and binds TWEAK, and an antibody that binds theTWEAK receptor.
 2. The method of claim 1 wherein the composition furthercomprises a pharmaceutically acceptable carrier.
 3. The method of claim1 wherein the mammal is a human.
 4. The method of claim 1 wherein themammal has a disease or condition mediated by angiogenesis.
 5. Themethod of claim 4 wherein the disease or condition is characterized byocular neovascularization.
 6. The method of claim 4 wherein the diseaseor condition is a malignant or metastatic condition.
 7. The method ofclaim 6 wherein the malignant or metastatic condition is a solid tumor.8. The method of claim 6 wherein the method further comprises treatingthe mammal with radiation.
 9. The method of claim 6 wherein the methodfurther comprises treating the mammal with a chemotherapeutic agent. 10.The method of claim 9 wherein the chemotherapeutic agent is selectedfrom the group consisting of alkylating agents, antimetabolites, vincaalkaloid, plant-derived chemotherapeutics, nitrosoureas, antitumorantibiotics, antitumor enzymes, topoisomerase inhibitors, platinumanalogs, adrenocortical suppressants, hormones, hormone agonists,hormone antagonists, antibodies, immunotherapeutics, blood cell factors,radiotherapeutics, and biological response modifiers.
 11. The method ofclaim 9 wherein the chemotherapeutic agent is selected from the groupconsisting of cisplatin, cyclophosphamide, mechloretamine, melphalan,bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine,methotrexate, taxol, asparaginase, vincristine, vinblastine,lymphokines, cytokines, interleukins, interferons, alpha interferon,beta interferon, delta interferon, TNF, chlorambucil, busulfan,carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine,mercaptopurine, thioguanine, vindesine, etoposide, teniposide,dactinomycin, daunorubicin, doxorubicin, plicamycin, mitomycin,L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, andfluoxymesterone.
 12. The method of claim 9 wherein the chemotherapeuticagent is selected from the group consisting of Flt3 ligand, CD40 ligand,interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNFantagonists, TNF receptor antagonists, TRAIL, CD148 agonists, VEGFantagonists, VEGF receptor antagonists, and Tek antagonists.
 13. Themethod of claim 1 wherein the antagonist comprises an antibody thatbinds specifically to the TWEAK receptor extracellular domain.
 14. Themethod of claim 13, wherein the antibody is selected from the groupconsisting of a monoclonal antibody, a humanized antibody, a transgenicantibody, and a human antibody.
 15. The method of claim 13 wherein theantibody is conjugated to a radioisotope, a plant-derived toxin, afungus-derived toxin, a bacterial-derived toxin, ricin A, diphtheriatoxin, or a chemical poison.
 16. The method of claim 13, wherein themammal has a disease or condition mediated by angiogenesis.
 17. Themethod of claim 16 wherein the disease or condition is characterized byocular neovascularization.
 18. The method of claim 16 wherein thedisease or condition is a malignant or metastatic condition.
 19. Themethod of claim 18 wherein the malignant or metastatic condition is asolid tumor.
 20. The method of claim 18 wherein the method furthercomprises treating the mammal with radiation.
 21. The method of claim 18wherein the method further comprises treating the mammal with achemotherapeutic agent.
 22. The method of claim 21 wherein thechemotherapeutic agent is selected from the group consisting ofalkylating agents, antimetabolites, vinca alkaloid, plant-derivedchemotherapeutics, nitrosoureas, antitumor antibiotics, antitumorenzymes, topoisomerase inhibitors, platinum analogs, adrenocorticalsuppressants, hormones, hormone agonists, hormone antagonists,antibodies, immunotherapeutics, blood cell factors, radiotherapeutics,and biological response modifiers.
 23. The method of claim 21 whereinthe chemotherapeutic agent is selected from the group consisting ofcisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin,carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol,asparaginase, vincristine, vinblastine, lymphokines, cytokines,interleukins, interferons, alpha interferon, beta interferon, deltainterferon, TNF, chlorambucil, busulfan, carmustine, lomustine,semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine,thioguanine, vindesine, etoposide, teniposide, dactinomycin,daunorubicin, doxorubicin, plicamycin, mitomycin, L-asparaginase,hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone.24. The method of claim 21 wherein the chemotherapeutic agent isselected from the group consisting of Flt3 ligand, CD40 ligand,interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNFantagonists, TNF receptor antagonists, TRAIL, CD148 agonists, VEGFantagonists, VEGF receptor antagonists, and Tek antagonists.
 25. Themethod of claim 1 wherein the antagonist disrupts the interactionbetween the TWEAK receptor and a TRAF molecule.